Despite recent advances in antiviral drug development, hepatitis C virus (HCV) remains a major cause of chronic liver disease and cancer, access to therapy is limited, drug resistance evolves rapidly, and there is no effective vaccine. Thus, there is an unmet need to improve therapy and to create a vaccine. As most antiviral vaccines elicit humoral responses against virus particles, further research is needed to understand the structure and assembly of HCV particles, which are protected from antibody neutralization through an as-yet-uncharacterized interaction with serum lipoproteins. HCV is also a valuable model system for understanding the Flaviviridae - a large family of clinically important viral pathogens - as well as positive-strand RNA viruses more generally. A key unanswered question is how do positive-strand RNA virus-encoded helicases contribute to virus assembly? This proposal builds upon our success in the previous funding period to examine HCV particle assembly, both as a unique paradigm of virus structure and as a model system for understanding helicase function. By leveraging our prior success and adapting new approaches, we will achieve the following three Aims: 1) establish the role of the HCV helicase domain in virus assembly; (2) define functional interactions within the virus assembly complex; and (3) determine the role of Apolipoprotein E in virus structure. Successful completion of this Project will lay the groundwork for understanding, at a deep mechanistic level, the role of viral helicases during the assembly of enveloped, positive-strand RNA viruses, provide insights into the coordination of nucleocapsid assembly, and - for the first time - rigorously test the prevailing model of HCV particle structure.